1. Field of the Invention
The present invention relates to a novel method for introducing a gene into a plant using genetic engineering techniques. Also, the present invention relates to a vector for introducing a gene into a plant used for the method.
2. Description of the Background
Transformation of microorganisms and cultured cells using genetic engineering is currently applied to the production of physiologically active substances useful as medicines and the like, and thus greatly contributes to the industry. In the field of plant breeding, industrial application of genetic engineering lags behind because the life cycles of plants are much longer than those of microorganisms and the like. However, since this technology enables a desired gene to be directly introduced into plants to be bred, it has the following advantages compared to classical breeding which requires multiple crossing: (a) it is possible to introduce only a characteristic to be improved; (b) it is possible to introduce characteristics of species other than plants (such microorganisms and the like); and (c) it is possible to greatly shorten the breeding period. Thus, a number of useful transgenic plants have been produced mainly in Europe and the U.S. and are now on the market.
Specifically, the production of transgenic plants requires the following three steps: (1) introducing the desired gene into the plant cell (including introduction of the same into the chromosomes, nucleus and the like); (2) selecting plant tissue made only of cells into which the desired gene has been introduced; and (3) regenerating a plant from the selected plant tissue. Among these steps, in selecting the desired transgenic tissue, generally, since it is difficult to confirm with the naked eye a tissue in which the desired gene is expressed (the tissue in which the desired gene is expressed is naturally a tissue constituted by cells into which the gene is introduced) without regenerating a plant, the desired gene is introduced into a plant cell together with a selectable marker gene of which expression can be easily detected at the stage of cell culturing, and the presence or absence of the expression of the selectable marker gene (namely, the presence or absence of the introduction of the selectable marker gene) is used as an index for the introduction of the desired gene. Examples of the selectable marker gene include a kanamycin-resistant gene (NPTII: neomycin phosphotransferase gene) and a hygromycin-resistant gene (hygromycin phosphotransferase gene) which impart resistance to antibiotics, a nopaline synthetase gene (NOS) and an octopine synthetase gene (OCS) which relate to amino acid synthesis, and a sulfonylurea-resistant gene (ALS: acetolactate synthetase gene) which imparts resistance to agricultural chemicals.
However, the expression of a selectable marker gene becomes a serious obstacle when its object is to apply such a transgenic plant to food. Namely, it is difficult to assure safety of the gene product obtained by the expression of the selectable marker gene on the human body. Consequently, when a transgenic plant produced using a selectable marker gene as an index is sold as food, it is necessary to carry out detailed examination on the effect of the gene product upon the human body. For example, although the NPTII gene has been already often used as a selectable marker gene at a laboratory level since the first half of 1980's, its gene product was approved by Food and Drug Administration (FDA) as a food additive for the first time in 1994, and transgenic plants to which the gene is introduced as a selectable marker gene have been used for food thereafter. However, uneasiness about such NPTII gene products is still present unavoidably at the essential level of consumers who actually eat these products.
Also, all of the genes which have so far been put into practical use as selectable marker genes, including the NPTII gene, are genes that contribute to the detoxication activity of plant cell growth inhibitors, so that selection of a tissue introduced with a desired gene is carried out by culturing the tissue using a medium containing such a growth inhibitor and evaluating the presence or absence of the expression of the selectable marker gene, namely resistance to the inhibitor, as an index. In that case, however, the presence of resistance, namely the ability of the plant tissue to grow in the presence of such an inhibitor, is merely a matter of degree, so that it is difficult to avoid undesirable influences of the culturing in the presence of such an inhibitor upon plant cells, and such influences are actually causing side effects, such as reduction of growth and redifferentiation ratio of the transgenic tissue due to decreased activity of the plant cells.
Furthermore, after selection of a transgenic tissue, expression of a selectable marker gene will cause considerable obstacles even at the level of researchers studying the plant breeding. That is, when a transgenic plant which has been produced by using a selectable marker gene is again introduced by another gene, introduction of the gene cannot be carried out using the same selectable-marker gene. In other words, since the selectable marker gene has been already present in the plant, the selectable marker gene is always expressed in the plant whether or not the new desired gene is introduced into the plant together with the selectable marker gene. Therefore, such a selectable marker gene can no longer be used as an index of the introduction of the new desired gene. Consequently, the number of times of repeated gene introducing into a certain plant is naturally restricted by the number of different selectable marker genes useful in the plant. However, kinds of selectable marker genes so far available are not so many. Additionally, all of the selectable marker genes are not necessarily useful in the plant of the object.
As a means for resolving these problems, the inventors of the present invention have previously provided a novel vector in International Publication No. WO 96/15252. This vector uses a morphological abnormality induction gene as a selectable marker gene which is present in plants in the natural world and whose safety upon the human body is secured to a certain degree. Additionally, when introduction of a gene into a plant is carried out using this vector, a transgenic tissue can be selected easily using its morphology as an index. That is, a tissue after a gene introduction treatment is cultured under appropriate conditions, and a tissue formed during the culturing showing abnormal morphology is detected and selected. It is not necessary to add an inhibitor which reduces plant cell activity to the medium during culturing. Also, when introduction of a gene into a plant is carried out using this vector in which the selectable marker gene is used in combination with a removable DNA element, a transgenic tissue from which influences of the selectable marker gene are completely removed can be obtained. Such a tissue can be obtained easily by merely carrying out its selection using morphology of the transgenic tissue as an index similar to the case of the above-described gene introduction.
However, even if gene introduction to a plant is carried out using such a vector, there is desire that improves working efficiency at the selection of the transgenic tissue.
That is, when a gene relating to production of a plant hormone is used as the morphological abnormality induction gene, the plant hormone produced by the gene expression in a transgenic cell migrates into its peripheral cells to provide influences indirectly, thus sometimes causing differentiation of adventitious shoots and adventitious roots from the nontransgenic cells which have received the influences. On the other hand, in the case of a tissue showing abnormal morphology generated from the transgenic cell which is directly influenced by the plant hormone, it is difficult in many cases to distinguish the abnormal morphology from normal adventitious shoots or roots at the initial stage of its generation. Accordingly, generally, the adventitious shoots or roots generated from the tissue after the gene introduction are separated and cultured, and then selection of the transgenic tissue is generally carried out. As a result of such culturing, morphological differences between a nontransgenic tissue and a transgenic tissue to such an extent that they can be distinguished with the naked eye, even in the case where these tissues are cultured as they are without separation. Then, the adventitious shoots and the like thus separated and cultured are actually occupied with the nontransgenic tissues at a marked ratio, and as a result, working efficiency at the selection of the transgenic tissue afterward is lowered.